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Corresponding author:
Zharaa B. Hassan
Article Citation:
Zharaa B. Hassan, Jasim M. Awada and Ahmed K. Hassan
Isolation and identification of Saccharomyces cerevisiae and evaluation of
biodegradation efficiency of acetamiprid during fermentation
Journal of Research in Ecology (2018) 6(2): 2097-2104
Dates:
Received: 19 Aug 2018 Accepted: 03 Sep 2018 Published: 22 Sep 2018
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Figure 3. HPLC spectra of acetamiprid biodegradation by S. cerevisiae YF (a) 15 ppm, (b) 10 ppm
flour, wheat dough was prepared using conventional technical material (99% purity) in 100 mL of HPLC
procedure according to (AACC, 2000) with some modi- grade acetonitrile. From this stock, intermediate stock
fication. The dough was kept for fermentation at room solutions of 4 ppm and 0.05 ppm were prepared which
temperature for 1 h. Finally, it was examined for degra- was shown in Figure 1.
dation of pesticide residues during the process by Extraction procedure
HPLC. Extraction of pesticide residue was accom-
Preparation of standards (Acetamiprid 20 SP) plished according to the QuEChERS method of Ana-
Acetamiprid technical material was obtained stassiades et al. (2003) with some modifications. 10 g of
from Dr. Ehrenstorfer GmbH, Germany. The stock solu- sample, homogenized and humidified, were weighed in
tion of 100 ppm was prepared by dissolving 0.01 g of a 50 mL centrifuge tube. 10 mL of acetonitrile, contain-
Table 3. Biodegradation of acetamiprid by ing 1 % (v/v) of acetic acid was added to the sample,
S. cerevisiae YF during wheat fermentation
and the mixture was vortexed for 1 min. After that, 3 g
Fortification level Residual
S. No of MgSO4 was added and vortexed immediately for 20
(ppm) concentration (ppm)
1 0.82 0.17 sec. Later, 1.7 g of sodium acetate and 0.5 g of disodi-
2 0.61 0.071 um hydrogen citrate sesquihydrate were added and the
3 0.17 0.044 tube was hand shaken for 1 min and centrifuged at 4000
4 0.14 0.058 x g for 8 min to provide a completely phased separation.
Figure 3. HPLC spectra of acetamiprid biodegradation by S. cerevisiae YF (c) 5 ppm, (d) Control
Finally, it was filtered by Buchner funnel to obtain a product of 845 bp, after showing the band in 1% aga-
clear supernatant, then further filtered by using 0.45 µm rose, visualized under UV and after staining with ethidi-
and transferred to vial before injection into HPLC fil- um bromide, as shown in Figure 2. PCR products were
tered by using 0.22 µm. sent to Macrogen/ Korean to determine the sequence of
The data were analyzed statistically and the re- the nitrogen bases. The ITS regions were easily ampli-
sults were expressed in means using Microsoft excel fied with universal primers that are compatible among
software. most fungal species. It has shown sufficient genetic var-
iability for identification at interspecies level and has
RESULTS AND DISCUSSION been adopted as the official standard barcoding region
Screening of the potential strain for fungi (Schoch et al., 2012). These sequences were
Cells (yeast) were counted by hemocytometer, compared with the available information on these genes
the results showed in the Table 2 that one of the isolates in the NCBI through the BLAST nucleotide search to
7
1.5*10 cell/mL is larger in number than other isolates, identify the isolates. The results of the identification of
the isolation was capable of growing on the medium fungal isolation were concordant with the DNA
which was selected for further investigation. sequences at 98 % of S. cerevisiae sequences available
Molecular (PCR) identification in the NCBI database (Gen Bank).
It was done through PCR technique using uni-
versal fungus-specific ITS1 and ITS4 rRNA with a
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